Plant Cell Reports
Plant Cell Reports (1983) 2:169-171
© Springer-Verlag 1983
Somatic Embryogenesis and Plant Regeneration in an Interspecific Hybrid of Oryza D. H. Ling, W. Y. Chen, M. F. Chen, and Z. R. Ma South China Institute of Botany, Academia Sinica, Guang-zhou, People's Republic of China Received January 26, 1983 / May 25, 1983 - Communicated by J. K. Vasil
Abstract
Materials
An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F I. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heartshape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.
Materials: The material was immature panicles of a specific hybrid F I (Oryza sativa (AA, 2n = 24) x O. latifolia (CCDD, 2n ~ . T - - - ~ g e n o t y p e of the h~brid is ACD, 2n = 36. The hybrid was produced by the Agricultural Scientific Academy in Guang-dong province, China in 1958, has not produced seed (sterile) since 1958 and is being maintained over 24 years vegetatively.
Abbreviations 2,4-D - 2,4-dichlorophenoxyacetic acid, NAA - naphthaleneacetic acid, BA - benzyladenine, IAA - indole acetic acid, kn - kinetin, MS - Murashige and Skoog medium (Murashige and Skoog 1962), HE - HE 5 medium (Ling et al. 1977), H - H medium (Nitsch 1969), N 6 N 6 medium (Zhu et al. 1975), B 5 - B 5 medium (Gamborg et al. 1968). Key words: Oryza, panicle culture
Somatic embryogenesis,
Immature
Introduction In the recent years, somatic embryogenesis and plantlets (via embryoids) have been successfully obtained in many important graminaceous crops such as in sugarcane (Zhen 1979), maize (Lu et al. 1982), sorghum (Brettell et al. 1980), wheat (Ozias-Akins and Vasil 1982), pearl millet (Vasil and Vasil 1980, 1981a, b and 1982), napier grass (Haydu and Vasil 1981 and Wang and Vasil 1982), Panicum maximum (Lu et al. 1981a, b, c and 1982a, b) and others from young embryos, immature panicles, and young leaves. Rice is an important cereal crop in which somatic embryogenesis has not been reported, although plantlets have been obtained from calli derived from different organs such as root (Kawata et al. 1968), shoot (Tsai et al. 1978), leaf sheath (Bhattacherya et al. 1980), immature panicle (Shu 1980), and seed embryo (Nakano 1975). In this communication, we report the somatic embryogenesis and plant regeneration from cultured immature panicles of a species hybrid of Oryza (2" sativa x O. latifolia).
and Methods
The stage of the immature panicle: The immature panicle (5-25 mm) from initiation of the second rachis to the formation of stamens and pistils were used as an explant. The suitable boots were separated and washed in running tap water. The outermost leaves and sheaths were peeled and cleaned with cotton soaked with 70% alcohol. The boots were continually peeled until the last sheath and node emerged, and then soaked in 75% alcohol for 3 minutes, rinsed 3 times in sterilized water followed by 0.1% mercuric chloride treatment for 10-15 minutes and washed 3 times in sterilized water. Later, the immature panicles were carefully dissected from the boot in petri dishes. Small callus pieces (2-4 mm) were transferred every 30 days for subculture and differentiation. The cultures were incubated in the dark at 28+2°C and plantlets were differentiated in 28+_2°C 12-T8 hr of light. The suitable embryogenic callus was fixed with both glutaraldehyde and osmium acid and then coated with gold and scanned by electron microscope. Medium: The different medium used for induction, subculture and differentiation of embryogenic callus are given in Table i. Results
and Discussion
Induction of embryogenic callus: After 7-10 days of inoculation, the immature panicles on HE medium showed swelling and initiation of ealli on the surface of the spikelets. The embryoids appeared on the slow growth callus after 2 months and new embryoids continuously arised on the old one. The embryogenic callus was compact and granulate in structure (Fig. i) and milk-white in colour. The callus included embryoids in various developmental stage, such as globular, heart-shape, scutellum-shape and mature embryoid and all of them could be easily separated from embryogenie callus (Fig. 3). The embryoids germinated immediately when they were transferred onto differentia' tion medium (Fig. 2). About 20-40 plantlets can be got in an average from a piece of callus (2-4 mm in size) after 30 days of transferring. The plantlets
170 Table I.
The supplementary
Media
elements
2,4-D
Callus
induction
HE
2
Callus
subculture
HE MS H N6 B5
2 2 2 0.4 0.4
HE MS N6 B5
-
Plant differentiation
Fig. 2. Germinating embryoid formed a new one (arrows), electron microscope scanning (x 20)
(mg/l).
NAA
kn
YE
2
3
1360
300
6
3 ,
1360 -
300 -
6 3 3 3 2
-
6 3 3 2
2 IAA 0.2 -
grew very well in the soil until mature and they are sterile also (Fig. 4). Lu et al. (1982) found that a combination of 0.5 mg/l 2,4-D with high concentration of sucrose (12%) was the most suitable for formation of embryogenic callus in corn. Kao et al. (1981) obtained embryogenesis in several species of alfalfa and pointed out that a combination of 2,4-D (or NAA) with cytokinin (zeatin-riboside or BA) were essential for embryoid induction and the concentration of these compounds was critical for embryogenesis. Narayanaswamy (1977) also suggested that casein hydrolyzate and yeast extract facilitated formation of embryoid. It appears that high concentration of sucrose and combination of auxin with cytokinin as well as presence of casein hydrolyzate and yeast extract would be favourable in somatic embryogenesis in somatic culture.
Fig. I. Scanning photograph of embryogenic callus in rice x 20.
of different media
0.5 1
2 2 2 2
2 2 2 2
embryogenesis
-
CH
Sucrose
(%)
in rice.
Proliferation and differentiation of embryogenic callus___: The embryogenic callus could proliferate on all five subculture media (Table 2), however, the subculture media have different effects on differentiation. The frequency of differentiation of the callus subcultured on H medium was the lowest and those subcultured on HE and MS media were high. The result in Table 2 showed HE and MS media to be most suitable for subculture and MS for high frequency of differentiation of the embryogenic callus. Interestingly, the embryogenic callus has successfully been subcultured for 12 months and the differentiation frequency was more than 80% in each passage (Table 3). The effect of duration of continuous subculture on the ability to proliferate and differentiate was stud-
Fig. 3. Embryoids of various developmental stage separating from an embryogenic callus, G: globular shape, H: heart shape, S: scutellum shape embryoid
Fig. 4. A mature plantlet derived from embryogenesis.
(x 4o). The HE medium has a high concentration of sucrose (6%), combination of auxin (2,4-D and NAA) with cytokinin (kinetin) as well as plenty of organic substance (casein hydrolyzate and yeast extract) (Ling et al. 1977). The medium was found to be quite suitable for somatic embryogenesis in this experiment. It is worth r e s e a r c h i n g further whether these three factors mentioned above are the major ones for inducing somatic
ied. Five durations of continuous subculture (i month, 2, 3, 4 and 5 months) were tested. The ability of the callus both to proliferate and to differentiate was most vigorous when they were subcultured after i and 2 months, respectively. In this experiment, we screened successfully HE, HE or MS and MS medium for induction, proliferation and differentiation separately. This experiment shows
171 Table 2.
Effect of different media on differentiation of embryogenic calli in subculture.
Subculture medium Differentiation medium
1
B5 2
HE
22
13
59.1
23
4
17.4
20
MS
22
13
59.1
22
6 (I)*
27.3
21
21
2
9.5
%
N6 B5
22
16
H 2
1
72.7
1 - number of calli transferred,
%
-
MS 2
1
7 (2)*
%
HE 2
1
%
35.0
21
18
85.7
18
15 (i)*
83.3
21
14 (i)*
66.7
18
12 (I)*
66.7
26
25
96.2
29
22
75.8
2 - number of calli with plantlets,
4
19.05
% - T2 x I00.
* The numberals in the bracket refer to the number of albino. Table 3. Subculture passages Month
The effect of
subculture times on ability to differentiation.
1
2
3
4
5
6
7
8
9
i0
ii
12
Mar
Apr
May
June
1982 July
Aug
Sept
Oct
Nov
Jan
1983 Feb
Mar
21
20
22
21
22
25
36
20
24
24
20
26
No. of calli with plantlet
18
16
18
19
18
21
29
16
19
23
16
22
%
85.7
80.0
81.8
90.5
81.8
84.0
80.5
80.0
79.2
95.8
80.8
84.6
No. of calli transferred
that induction, proliferation and differentiation of embryogenic callus are the three key steps for establishing system of somatic embryogenesis in rice.
Lu, C., Vasil, I.K. (1982) Amer. J. Bot° 69: 77-81. Lu, C., Vasil, I.K., Ozias-Akins, P. (1982) Theor. Appl. Genet. 62:109-112.
Acknowledgements We thank Dr. D.S. Brar for review and correction of the manuscript. References Bhattacherya, P., Sen, S.K. (1980a) Theor. Appl. Genet. 56:87-90.
Murashige, T., F. Skoog (1962) Physiol. Plant 15:473497. Nakano, H., Tashiro, T., Maeda, E. (1975) Z. Pflanzenphysiol. 76:444-449. Narayanaswamy, S. (1977) In Applied and Fundamental Aspects Of Plant Cell, Tissue and Organ Culture. (Reinert and Bajaj eds.) Springer-Verlag, 179-248.
Brettell, R.I.S., Wernicke, W., Thomas, E. (i980) Protoplasma 104:141-148.
Nitsch, J.P. (1969) Phytomorphol. 19:389-404.
Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu, C.Y. (1975) Sci. Sin. 16:659-688.
Ozias-Akins, P., Vasil, I.K. (1982) Protoplasma ii0: 95-105.
Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Exp. Cell Res. 50:151-158.
Shu, L.H., Wei, J.Y. (1980) Acta Wuhan University Jour. No. 1:94-100.
Haydu, Z., Vasil, K. (1981) Theor. Appl. Genet. 59: 269-273.
Tsai, Y.X. (1978) Sci. Press, Peking.
pp. 517-519.
Kao, K.N., Michayluk, M.R. (1981) In Vitro 17:645-648.
Vasil, I.V., Vasil, I°K. (1980) Theor. Appl. Genet. 56:97-99.
Kawata, S., Ishihara, A. (1968) Proc. Japan Acad. 44: 549-552.
Vasil, V., Vasil, I.K. (1981a) Amer. Jour. Bot. 68: 864-872.
Ling, D.H., Xian, W.N., Zeng, B.L. (1977) Proc. Symp. on Anther Culture, Sci. Press, Peking. pp. 265.
Vasil, V., Vasil, I.K. (1981b) Ann. Bot. 47:447-478.
Lu, C., Vasil, V., Vasil, I.K. (1981a) Z. Pflanzenphysiol. 104:311-318.
Vasil, V., Vasil, I.K. (1982) Amer. Jour. Bot. 69: 1441-1449.
Lu, C., Vasil, I.K. (1981b) Ann. Bot. 47:543-548.
Wang, D., Vasil, I.K. (1982) Plant Sci. Lett. 25:147 154.
Lu, C., Vasil, I.K. (1981c) Theor. Appl. Genet. 59: 275-280.
Zheng, J.S. (1979) Acta Phytophysiol. Sinica 5:411416.
Plant Cell Reports (1983) 2:169-171
© Springer-Verlag 1983
Somatic Embryogenesis and Plant Regeneration in an Interspecific Hybrid of Oryza D. H. Ling, W. Y. Chen, M. F. Chen, and Z. R. Ma South China Institute of Botany, Academia Sinica, Guang-zhou, People's Republic of China Received January 26, 1983 / May 25, 1983 - Communicated by J. K. Vasil
Abstract
Materials
An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F I. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heartshape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.
Materials: The material was immature panicles of a specific hybrid F I (Oryza sativa (AA, 2n = 24) x O. latifolia (CCDD, 2n ~ . T - - - ~ g e n o t y p e of the h~brid is ACD, 2n = 36. The hybrid was produced by the Agricultural Scientific Academy in Guang-dong province, China in 1958, has not produced seed (sterile) since 1958 and is being maintained over 24 years vegetatively.
Abbreviations 2,4-D - 2,4-dichlorophenoxyacetic acid, NAA - naphthaleneacetic acid, BA - benzyladenine, IAA - indole acetic acid, kn - kinetin, MS - Murashige and Skoog medium (Murashige and Skoog 1962), HE - HE 5 medium (Ling et al. 1977), H - H medium (Nitsch 1969), N 6 N 6 medium (Zhu et al. 1975), B 5 - B 5 medium (Gamborg et al. 1968). Key words: Oryza, panicle culture
Somatic embryogenesis,
Immature
Introduction In the recent years, somatic embryogenesis and plantlets (via embryoids) have been successfully obtained in many important graminaceous crops such as in sugarcane (Zhen 1979), maize (Lu et al. 1982), sorghum (Brettell et al. 1980), wheat (Ozias-Akins and Vasil 1982), pearl millet (Vasil and Vasil 1980, 1981a, b and 1982), napier grass (Haydu and Vasil 1981 and Wang and Vasil 1982), Panicum maximum (Lu et al. 1981a, b, c and 1982a, b) and others from young embryos, immature panicles, and young leaves. Rice is an important cereal crop in which somatic embryogenesis has not been reported, although plantlets have been obtained from calli derived from different organs such as root (Kawata et al. 1968), shoot (Tsai et al. 1978), leaf sheath (Bhattacherya et al. 1980), immature panicle (Shu 1980), and seed embryo (Nakano 1975). In this communication, we report the somatic embryogenesis and plant regeneration from cultured immature panicles of a species hybrid of Oryza (2" sativa x O. latifolia).
and Methods
The stage of the immature panicle: The immature panicle (5-25 mm) from initiation of the second rachis to the formation of stamens and pistils were used as an explant. The suitable boots were separated and washed in running tap water. The outermost leaves and sheaths were peeled and cleaned with cotton soaked with 70% alcohol. The boots were continually peeled until the last sheath and node emerged, and then soaked in 75% alcohol for 3 minutes, rinsed 3 times in sterilized water followed by 0.1% mercuric chloride treatment for 10-15 minutes and washed 3 times in sterilized water. Later, the immature panicles were carefully dissected from the boot in petri dishes. Small callus pieces (2-4 mm) were transferred every 30 days for subculture and differentiation. The cultures were incubated in the dark at 28+2°C and plantlets were differentiated in 28+_2°C 12-T8 hr of light. The suitable embryogenic callus was fixed with both glutaraldehyde and osmium acid and then coated with gold and scanned by electron microscope. Medium: The different medium used for induction, subculture and differentiation of embryogenic callus are given in Table i. Results
and Discussion
Induction of embryogenic callus: After 7-10 days of inoculation, the immature panicles on HE medium showed swelling and initiation of ealli on the surface of the spikelets. The embryoids appeared on the slow growth callus after 2 months and new embryoids continuously arised on the old one. The embryogenic callus was compact and granulate in structure (Fig. i) and milk-white in colour. The callus included embryoids in various developmental stage, such as globular, heart-shape, scutellum-shape and mature embryoid and all of them could be easily separated from embryogenie callus (Fig. 3). The embryoids germinated immediately when they were transferred onto differentia' tion medium (Fig. 2). About 20-40 plantlets can be got in an average from a piece of callus (2-4 mm in size) after 30 days of transferring. The plantlets
170 Table I.
The supplementary
Media
elements
2,4-D
Callus
induction
HE
2
Callus
subculture
HE MS H N6 B5
2 2 2 0.4 0.4
HE MS N6 B5
-
Plant differentiation
Fig. 2. Germinating embryoid formed a new one (arrows), electron microscope scanning (x 20)
(mg/l).
NAA
kn
YE
2
3
1360
300
6
3 ,
1360 -
300 -
6 3 3 3 2
-
6 3 3 2
2 IAA 0.2 -
grew very well in the soil until mature and they are sterile also (Fig. 4). Lu et al. (1982) found that a combination of 0.5 mg/l 2,4-D with high concentration of sucrose (12%) was the most suitable for formation of embryogenic callus in corn. Kao et al. (1981) obtained embryogenesis in several species of alfalfa and pointed out that a combination of 2,4-D (or NAA) with cytokinin (zeatin-riboside or BA) were essential for embryoid induction and the concentration of these compounds was critical for embryogenesis. Narayanaswamy (1977) also suggested that casein hydrolyzate and yeast extract facilitated formation of embryoid. It appears that high concentration of sucrose and combination of auxin with cytokinin as well as presence of casein hydrolyzate and yeast extract would be favourable in somatic embryogenesis in somatic culture.
Fig. I. Scanning photograph of embryogenic callus in rice x 20.
of different media
0.5 1
2 2 2 2
2 2 2 2
embryogenesis
-
CH
Sucrose
(%)
in rice.
Proliferation and differentiation of embryogenic callus___: The embryogenic callus could proliferate on all five subculture media (Table 2), however, the subculture media have different effects on differentiation. The frequency of differentiation of the callus subcultured on H medium was the lowest and those subcultured on HE and MS media were high. The result in Table 2 showed HE and MS media to be most suitable for subculture and MS for high frequency of differentiation of the embryogenic callus. Interestingly, the embryogenic callus has successfully been subcultured for 12 months and the differentiation frequency was more than 80% in each passage (Table 3). The effect of duration of continuous subculture on the ability to proliferate and differentiate was stud-
Fig. 3. Embryoids of various developmental stage separating from an embryogenic callus, G: globular shape, H: heart shape, S: scutellum shape embryoid
Fig. 4. A mature plantlet derived from embryogenesis.
(x 4o). The HE medium has a high concentration of sucrose (6%), combination of auxin (2,4-D and NAA) with cytokinin (kinetin) as well as plenty of organic substance (casein hydrolyzate and yeast extract) (Ling et al. 1977). The medium was found to be quite suitable for somatic embryogenesis in this experiment. It is worth r e s e a r c h i n g further whether these three factors mentioned above are the major ones for inducing somatic
ied. Five durations of continuous subculture (i month, 2, 3, 4 and 5 months) were tested. The ability of the callus both to proliferate and to differentiate was most vigorous when they were subcultured after i and 2 months, respectively. In this experiment, we screened successfully HE, HE or MS and MS medium for induction, proliferation and differentiation separately. This experiment shows
171 Table 2.
Effect of different media on differentiation of embryogenic calli in subculture.
Subculture medium Differentiation medium
1
B5 2
HE
22
13
59.1
23
4
17.4
20
MS
22
13
59.1
22
6 (I)*
27.3
21
21
2
9.5
%
N6 B5
22
16
H 2
1
72.7
1 - number of calli transferred,
%
-
MS 2
1
7 (2)*
%
HE 2
1
%
35.0
21
18
85.7
18
15 (i)*
83.3
21
14 (i)*
66.7
18
12 (I)*
66.7
26
25
96.2
29
22
75.8
2 - number of calli with plantlets,
4
19.05
% - T2 x I00.
* The numberals in the bracket refer to the number of albino. Table 3. Subculture passages Month
The effect of
subculture times on ability to differentiation.
1
2
3
4
5
6
7
8
9
i0
ii
12
Mar
Apr
May
June
1982 July
Aug
Sept
Oct
Nov
Jan
1983 Feb
Mar
21
20
22
21
22
25
36
20
24
24
20
26
No. of calli with plantlet
18
16
18
19
18
21
29
16
19
23
16
22
%
85.7
80.0
81.8
90.5
81.8
84.0
80.5
80.0
79.2
95.8
80.8
84.6
No. of calli transferred
that induction, proliferation and differentiation of embryogenic callus are the three key steps for establishing system of somatic embryogenesis in rice.
Lu, C., Vasil, I.K. (1982) Amer. J. Bot° 69: 77-81. Lu, C., Vasil, I.K., Ozias-Akins, P. (1982) Theor. Appl. Genet. 62:109-112.
Acknowledgements We thank Dr. D.S. Brar for review and correction of the manuscript. References Bhattacherya, P., Sen, S.K. (1980a) Theor. Appl. Genet. 56:87-90.
Murashige, T., F. Skoog (1962) Physiol. Plant 15:473497. Nakano, H., Tashiro, T., Maeda, E. (1975) Z. Pflanzenphysiol. 76:444-449. Narayanaswamy, S. (1977) In Applied and Fundamental Aspects Of Plant Cell, Tissue and Organ Culture. (Reinert and Bajaj eds.) Springer-Verlag, 179-248.
Brettell, R.I.S., Wernicke, W., Thomas, E. (i980) Protoplasma 104:141-148.
Nitsch, J.P. (1969) Phytomorphol. 19:389-404.
Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu, C.Y. (1975) Sci. Sin. 16:659-688.
Ozias-Akins, P., Vasil, I.K. (1982) Protoplasma ii0: 95-105.
Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Exp. Cell Res. 50:151-158.
Shu, L.H., Wei, J.Y. (1980) Acta Wuhan University Jour. No. 1:94-100.
Haydu, Z., Vasil, K. (1981) Theor. Appl. Genet. 59: 269-273.
Tsai, Y.X. (1978) Sci. Press, Peking.
pp. 517-519.
Kao, K.N., Michayluk, M.R. (1981) In Vitro 17:645-648.
Vasil, I.V., Vasil, I°K. (1980) Theor. Appl. Genet. 56:97-99.
Kawata, S., Ishihara, A. (1968) Proc. Japan Acad. 44: 549-552.
Vasil, V., Vasil, I.K. (1981a) Amer. Jour. Bot. 68: 864-872.
Ling, D.H., Xian, W.N., Zeng, B.L. (1977) Proc. Symp. on Anther Culture, Sci. Press, Peking. pp. 265.
Vasil, V., Vasil, I.K. (1981b) Ann. Bot. 47:447-478.
Lu, C., Vasil, V., Vasil, I.K. (1981a) Z. Pflanzenphysiol. 104:311-318.
Vasil, V., Vasil, I.K. (1982) Amer. Jour. Bot. 69: 1441-1449.
Lu, C., Vasil, I.K. (1981b) Ann. Bot. 47:543-548.
Wang, D., Vasil, I.K. (1982) Plant Sci. Lett. 25:147 154.
Lu, C., Vasil, I.K. (1981c) Theor. Appl. Genet. 59: 275-280.
Zheng, J.S. (1979) Acta Phytophysiol. Sinica 5:411416.
